Pharmaceutical Biology, 2010; 48(7): 753756

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Antimicrobial activity of crude epicarp and seed

extracts from mature avocado fruit (Persea americana)
of three cultivars
Teck Wah Raymond Chia 1,2, and Gary A. Dykes1,2
School of Land and Food Sciences, University of Queensland, Brisbane, Queensland, Australia, and 2Food Science
Australia, Tingalpa DC, Queensland, Australia

Abstract
The epicarp and seed of Persea Americana Mill. var. Hass (Lauraceae), Persea Americana Mill. var. Shepard, and
Persea americana Mill. var Fuerte cultivars of mature avocados (n=3) were ground separately and extracted
with both absolute ethanol and distilled water. Extracts were analyzed for antimicrobial activity using the
microtiter broth microdilution assay against four Gram-positive bacteria, six Gram-negative bacteria, and
one yeast. Antimicrobial activity against two molds was determined by the hole plate method. The ethanol
extracts showed antimicrobial activity (104.2416.7 g/mL) toward both Gram-positive and Gram-negative
bacteria (except Escherichia coli), while inhibition of the water extracts was only observed for Listeria monocytogenes (93.8375.0 g/mL) and Staphylococcus epidermidis (354.2 g/mL). The minimum concentration
required to inhibit Zygosaccharomyces bailii was 500 g/mL for the ethanol extracts, while no inhibition
was observed for the water extracts. No inhibition by either ethanol or water extracts was observed against
Penicillium spp. and Aspergillus flavus.
Keywords: Antimicrobial; avocado cultivar; epicarp; extracts; seed

Introduction
As the world population grows the demand for food
increases (Food and Agriculture Organization of the
United Nations, 2002), and there is therefore a need not
only to produce more food, but to assure that what is produced is safe for human consumption. One of the most
common factors contributing to unsafe food is bacterial
contamination (Conte et al., 2007). Synthetic additives
have been widely used in the food industry to control
microbial pathogens and inhibit microbial spoilage,
although in recent years there is an increasing demand
for natural food additives such as plant extracts (Conte
etal., 2007).
Plant materials such as Citrus spp. peel (Johann etal.,
2007) and grape (Vitis vinifera L.) seeds (Baydar etal.,
2006) are some natural products that display antimicrobial activity that has been applied in foods. The avocado

fruit [Persea Americana Mill. (Lauraceae)], a native

of tropical America (Jacob et al., 1971), is commonly
grown in many developing countries for the flesh of its
fruit (Chanderbali etal., 2008). Three botanical varieties
of avocado adapted to different climate conditions have
traditionally been recognized: Mexican [P. americana
var. drymifolia (Schlecht. & Cham.) Blake], Guatemalan
(P. americana var. guatemalensis L. Wms.), and West
Indian (P. americana var. americana Mill.). Most commercial avocado cultivars are interracial hybrids developed from chance seedlings. Thus, the most important
cultivars in subtropical climates, such as Hass, Bacon,
and Fuerte, are GuatemalanMexican hybrids with different degrees of hybridization (Newett etal., 2002).
Extracts from the epicarp of the immature avocado
fruit have been demonstrated to have both antifungal and
antibacterial properties (Jacob etal., 1971; Sivanathan &
Adikaram, 1989). The seed of the immature fruit was also

Address for Correspondence: Teck Wah Raymond Chia, Food Science Australia, PO Box 3312, Tingalpa DC, Queensland, Australia, 4173. Tel: +61 7 3214 2047.
Fax: +61 7 3214 2051. E-mail: Raymond.Chia@csiro.au
(Received 08 December 2008; revised 27 February 2009; accepted 07 June 2009)
ISSN 1388-0209 print/ISSN 1744-5116 online 2010 Informa UK Ltd
DOI: 10.3109/13880200903273922

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754 Teck Wah Raymond Chia and Gary A. Dykes

found to have antibacterial properties (Jacob etal., 1971).
The antifungal properties of the immature avocado were
established to be due to the idioblast oil cells, which are
made up of alkaloids, sesquiterpene hydroperoxides,
other terpenes (Platt & Thomson, 1992), persin, and a
group of 2-alkylfurans (Rodriguez-Saona etal., 1998).
Tannins, catechin flavones, and polyphenolic compounds are often found in the tissues and seed of the
avocado fruit. These chemicals are all antimicrobial in
nature and could have contributed to the antibacterial
activity of the immature fruit (Jacob et al. 1971; Young
& Biale, 1967). However, few data are available on the
antimicrobials of mature fruit, which are expected to be
different, as protection from plant pathogenic microorganisms is no longer required and microbial degradation
may even be desirable to release the seed.
Mature avocado flesh is often used to produce
guacamole or oil, leaving the epicarp and seed as a
byproduct. The potential use of the discarded epicarp
and seed could add value to fruit production in developing countries, and also potentially reduce environmental
problems associated with disposal of the epicarp and
seed. This study was therefore undertaken to determine
the presence and level of antimicrobial activity (if any)
associated with simple crude extracts of the epicarp
and seed of different varieties of mature avocados
against 10 foodborne pathogens and three fungi of food
significance.

Materials and methods

Preparation of avocado extracts
Hass (Guatemalan race), Shepard (Guatemalan race),
and Fuerte (Guatemalan Mexican race) cultivars of
avocado, selected due to their genetic differences (Newett
etal., 2002), were obtained from local suppliers between
February and May 2004 and kept matured until ready-toeat. The epicarp and seed of each individual avocado fruit
were retained and ground separately. Voucher specimens
were not deposited anywhere, as the fruit is freely available commercially.
Ethanol and water extracts were each prepared from
three avocados of each variety. Ethanol extracts of the
ground epicarp and seed were prepared by stirring 10g
of the homogenates in 50mL of absolute ethanol at 4C
for 24h (Emeruwa, 1982; Ulate-Rodriguez et al., 1997).
Extracts were recovered by filtration and dried in a rotary
evaporator at 70C for 15min. After drying, extracts were
weighed, reconstituted in 5mL of 50% ethanol, and stored
at room temperature until use.
Water extracts were prepared as above, but after filtration, the slurries were centrifuged at 10,000 g for 15min
after which the supernatants were decanted. The resulting

liquid was filtered and the filtered supernatants were

freeze-dried. The dried extracts were weighed, reconstituted in 5mL of distilled water, and then autoclaved. After
sterilization, the extracts were stored at room temperature until use (Richter & Vore, 1989).
Microorganisms used
Ten bacteria, Listeria monocytogenes (ATCC 7644),
Staphylococcus epidermidis (ATCC 12228), Staphylococcus
aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212),
Escherichia coli (ATCC 25922), Salmonella Enteritidis
(ATCC 13076), Citrobacter freundii (ATCC 8090),
Pseudomonas aeruginosa (ATCC 27853), Salmonella
Typhimurium (ATCC 13311), and Enterobacter aerogenes (ATCC 13048), and three fungi, Aspergillus flavus,
Penicillium spp., and Zygosaccharomyces bailii, from the
Food Microbiology Laboratory collection at the University
of Queensland were used.
Antibacterial activity determinations
The antibacterial activity of the extracts was determined
using the microtiter broth microdilution assay (CLSI,
2006, 2008). Briefly, 100 L of tryptic yeast soy glucose
broth was dispensed into all wells of a 96-well microtiter
plate. A diluted 1000 g/mL solution of each extract
(100L) was added to a separate well in the first column
of the microtiter plate. Serial two-fold dilutions were
made with a multichannel pipette, beginning with the
first column, and proceeding until the following concentrations of each antimicrobial agent were obtained:
500, 250, 125, 62.5, 31.25 and 15.625 g/mL. One hundred microliters of the contents of the sixth column
were discarded, and columns seven and eight were left
free of antimicrobials. Five microliters of an overnight
culture grown in nutrient broth at 37C containing ~108
109 cfu/mL was added to each well of the first seven columns. The wells in column eight were not inoculated and
served as a negative control. Streptomycin was used as
a positive control against these bacteria as described by
Amarowicz etal. (2008). Plates were incubated at 37C for
48h. Plates were inspected visually for growth at 24 and
48h. In this study, the minimum inhibitory concentration
(MIC) was taken as the lowest concentration of antimicrobial agent at which there was no perceptible growth
of the organism. Extracts with MICs equal to or greater
than 500 g/mL are described as having no antibacterial
activity. All antibacterial activity studies were performed
using three individual avocados of each variety.
Antifungal activity determinations
The antifungal activity of the extracts against the yeast
Zygosaccharomyces bailii was determined as for the

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Antimicrobial activity of avocado 755

antibacterial activity determinations described above. The
antifungal activity of the extracts against the two molds
was determined by the hole plate method using potato
dextrose agar (PDA) as the growth medium (Qamar etal.,
1996). Briefly, sterilized PDA inoculated with each of the
fungi was vortexed and aseptically poured into sterile
90mm Petri plates and allowed to congeal. Five holes of
0.6cm diameter were aseptically punched into the Petri
plates with a stainless steel borer of uniform edge and
size. The holes were filled with 100 L of epicarp or seed
extract from the three different cultivars of avocado at a
concentration of 500 g/mL. There were two negative
controls for each extract, one containing ethanol and
the other containing sterile distilled water. Petri plates
were incubated at 30C for up to 5 days. Clear zones of
inhibition around the holes were recorded as having antifungal activity. All antifungal activities were performed in
triplicate as above.
Statistical analysis
MannWhitney, KruskalWallis, and t tests were
performed on all data sets using MINITAB software
(MINITAB 15; Minitab Inc., Minneapolis, MN, USA) at a
95% confidence level.

Results and discussion

The results of the determination of antimicrobial activity
for all ethanol extracts of avocado against both bacterial

and fungal species are presented as MICs in Tables 1 and

2, respectively. The values presented for the bacteria and
yeast are those obtained after 24h (as these did not differ from those obtained at 48h). Gram-positive bacteria
that were susceptible to water extracts of avocado were
Listeria monocytogenes and Staphylococcus epidermidis (data not shown). Antimicrobial activity against
Listeria monocytogenes for water extracts ranged from
93.8 g/mL for the Shepard variety epicarp to 375.0 g/mL
for the seed of Fuerte variety. The only water extract that
displayed any activity against Staphylococcus epidermidis
was the epicarp of the Hass variety (354.2 g/mL). No
antibacterial activities were observed against the other
seven bacteria tested (Table 1).
In general the ethanol extracts displayed a wider
range and higher level of activity than the water extracts,
even though the results were not significantly different
(p>0.05). Ethanol extracts of avocado (from individual
epicarp, seed, and cultivar combinations) displayed activity against most Gram-positive (except Staphylococcus
epidermidis) and Gram-negative (except Escherichia
coli) bacteria tested (Table 1). Minimum inhibitory concentration values ranging from 104.2 g/mL (Salmonella
Enteritidis) to 416.7 g/mL (Listeria monocytogenes and
Staphylococcus aureus) were observed.
Water extracts of avocado (from specific epicarp, seed,
and cultivar combinations) displayed no activity against
any of the fungi tested, while MICs as low as 166.7 g/mL
from the corresponding ethanol extracts were observed
against Zygosaccharomyces bailii only (Table 2). It has
been speculated that as avocados mature, the degree of

Table 1. Antibacterial activity (minimum inhibitory concentration, g/mL) of ethanol extracts of three varieties of avocado against 10 bacteriaa.
Shepard
Hass
Fuerte
Epicarp
Seed
Epicarp
Seed
Epicarp
Seed
Listeria monocytogenes
416.7144.3
166.772.2
>500
>500
416.7144.3
125.00.0
Staphylococcus epidermidis
>500
>500
>500
>500
>500
>500
Staphylococcus aureus
416.7144.3
416.7144.3
291.7190.9
>500
416.7144.3
208.372.2
Enterococcus faecalis
>500
250.00.0
>500
>500
500.00.0
500.00.0
Escherichia coli
>500
>500
>500
>500
>500
>500
Salmonella Enteritidis
208.3252.6
208.3252.6
104.236.1
125.00.0
125.0108.3
145.895.5
250.0216.5
Citrobacter freundii
166.772.2
145.895.5
208.372.2
166.772.2
250.0216.5
Pseudomonas aeruginosa
166.772.2
166.772.2
208.372.2
250.0216.5
250.0216.5
291.7190.9
Salmonella Typhimurium
375.0216.5
250.0216.5
>500
>500
>500
>500
Enterobacter aerogenes
250.0216.5
125.00.0
250.0216.5
125.00.0
>500
>500
a
Data are means standard deviations of three individual avocados for each variety.

Table 2. Antifungal activity (minimum inhibitory concentration, g/mL) of ethanol extracts of three cultivars of avocado against one yeast and
two moldsa.
Shepard
Hass
Fuerte
Epicarp
Seed
Epicarp
Seed
Epicarp
Seed
375.0216.5
Zygosaccharomyces bailii
416.7144.3
416.7144.3
375.0216.5
104.236.1
166.772.2
Penicillium spp.
>500
>500
>500
>500
>500
>500
Aspergillus flavus
>500
>500
>500
>500
>500
>500
a
Data are means standard deviations of three individual avocados for each variety.

756 Teck Wah Raymond Chia and Gary A. Dykes

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antifungal activity decreases due to the breakdown of the

active ingredients (Karni etal., 1988).
The evidence presented in this article indicates that
crude extracts of the epicarp and seed of mature avocados
do have antimicrobials, and possess the potential to be
used as a food additive. Furthermore, this may represent
an alternative source of income from avocado waste. In
order to realize this potential, however, further studies
need to be performed to identify the active compounds in
the mature fruit and optimize their extraction on a larger
scale.

Declaration of interest
The authors report no conflicts of interest. The authors
alone are responsible for the content and writing of the
paper.

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